T Cell Responsiveness And Homeostasis In Anti-Tumor

Thomas F Gajewski, Associate Professor
University Of Chicago 5801 S Ellis Ave Chicago, Il 60637

Grant 5R01CA118153-02 from National Cancer Institute, IRG: TTT

Abstract: The progressive growth of antigen-expressing tumors despite the presence of tumor antigen-specific CD8+ T cells is a central problem in the field of anti-tumor immunity. Although one hypothetical mechanism of escape is T cell anergy, this process in the tumor setting is poorly defined. Using a TCP transgenic T cell model that allows recovery of sufficient numbers of T cells for analysis ex vivo, we have recently observed that tumors do induce antigen-specific hyporesponsiveness of anti-tumor CD8+ T cells and not of irrelevant T cells, consistent with a form of T cell anergy. We also have obtained preliminary data that tumor-induced T cell hyporesponsiveness does not occur when lymphopenia-induced homeostatic proliferation is permitted. Understanding the mechanism of the induction and reversal of T cell anergy should have great impact in our ability to maintain the functionality of tumor antigen-specific T cells in vivo, and enable the development of interventions that can be translated to the clinic. The first specific aim is to determine the mechanism of T cell-intrinsic hyporesponsiveness of TCR transgenic T cells in tumor bearing mice. TCR Tg T cells will be analyzed ex vivo for deficiencies in signal transduction events triggered by TCR/CD28 stimulation, and for molecular alterations using gene expression profiling. CAR Tg T cells and adenoviral vectors will be utilized to examine the causal relationship between observed changes and T cell dysfunction. The second specific aim is to explore and understand the mechanism by which T cell responsiveness is restored through homeostatic proliferation in vivo. Homeostatic signals will be delivered to anergic T cells by manipulating the host (transferring into RAG2-/- mice), manipulating the tumor (transfecting to express IL-7 and/or IL-15), or by manipulating the T cells (transduction with constitutively active Stat5). T cells will be analyzed for functional and biochemical properties, and alterations will be correlated with tumor rejection capability. The third specific aim is to apply knowledge of maintenance of T cell responsiveness out of this reductionist model toward rejection of established tumors in normal mice. T cell subsets from normal C57BL76 mice will be manipulated to affect homeostatic factors and tested for rejection of B16 melanoma transfected to express the model antigen SIY. T cell transduction and tumor transfectants will be examined using factors identified to be useful from the TCR Tg model and mechanisms of improved tumor control will be dissected.

Keywords: cell, homeostasis, neoplasm /cancer, Carnivora, anergy, antigen, complement, concept, dissection, emotion, gene expression profiling, immunity, lymphopenia, melanoma, model, suppression, tumor antigen

Project start date: 2006-09-28

Project end date: 2011-07-31

5R01CA118153-02 (2007): $261011

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T Cell Responsiveness And Homeostasis In Anti-Tumor

Thomas F Gajewski, Associate Professor
Pathologyuniversity Of Chicago

Grant 5R01CA118153-03 from National Cancer Institute, IRG: TTT

Abstract: The progressive growth of antigen-expressing tumors despite the presence of tumor antigen-specific CD8+ T cells is a central problem in the field of anti-tumor immunity. Although one hypothetical mechanism of escape is T cell anergy, this process in the tumor setting is poorly defined. Using a TCP transgenic T cell model that allows recovery of sufficient numbers of T cells for analysis ex vivo, we have recently observed that tumors do induce antigen-specific hyporesponsiveness of anti-tumor CD8+ T cells and not of irrelevant T cells, consistent with a form of T cell anergy. We also have obtained preliminary data that tumor-induced T cell hyporesponsiveness does not occur when lymphopenia-induced homeostatic proliferation is permitted. Understanding the mechanism of the induction and reversal of T cell anergy should have great impact in our ability to maintain the functionality of tumor antigen-specific T cells in vivo, and enable the development of interventions that can be translated to the clinic. The first specific aim is to determine the mechanism of T cell-intrinsic hyporesponsiveness of TCR transgenic T cells in tumor bearing mice. TCR Tg T cells will be analyzed ex vivo for deficiencies in signal transduction events triggered by TCR/CD28 stimulation, and for molecular alterations using gene expression profiling. CAR Tg T cells and adenoviral vectors will be utilized to examine the causal relationship between observed changes and T cell dysfunction. The second specific aim is to explore and understand the mechanism by which T cell responsiveness is restored through homeostatic proliferation in vivo. Homeostatic signals will be delivered to anergic T cells by manipulating the host (transferring into RAG2-/- mice), manipulating the tumor (transfecting to express IL-7 and/or IL-15), or by manipulating the T cells (transduction with constitutively active Stat5). T cells will be analyzed for functional and biochemical properties, and alterations will be correlated with tumor rejection capability. The third specific aim is to apply knowledge of maintenance of T cell responsiveness out of this reductionist model toward rejection of established tumors in normal mice. T cell subsets from normal C57BL76 mice will be manipulated to affect homeostatic factors and tested for rejection of B16 melanoma transfected to express the model antigen SIY. T cell transduction and tumor transfectants will be examined using factors identified to be useful from the TCR Tg model and mechanisms of improved tumor control will be dissected

Keywords: cell, homeostasis, neoplasm /cancer Carnivora, anergy, antigen, complement, concept, dissection, emotion, gene expression profiling, immunity, lymphopenia, melanoma, model, suppression, tumor antigen

Project start date: 2006-09-28

Project end date: 2011-07-31

MAGE-3 PLUS IL-12 IMMUNIZATION IN METASTATIC MELANOMA

Thomas F Gajewski, Associate Professor
University Of Chicago 5801 S Ellis Ave Chicago, Il 60637

Grant 5M01RR000055-390709 from National Center For Research Resources, IRG:

Abstract: Tumor antigen-specific immunotherapy of melanoma patients will be investigated. The patients will be HLA-typed and their tumors biopsied to analyze for MAGE-3 expression. If eligible, PBMC will be isolated and pulsed with the relevant MAGE-3 peptide. These will be washed and injected subcutaneously into the patient, along with rhIL-12. Safely, immune parameters, and tumor response will be monitored.

Keywords: combination antineoplastic therapy, human therapy evaluation, interleukin 12, melanoma, metastasis, neoplasm /cancer chemotherapy, neoplasm /cancer immunotherapy, tumor antigen, clinical trial phase I, clinical trial phase II /III /IV, clinical research, human subject

Core--Human Immunologic Monitoring

Thomas F Gajewski, Associate Professor
University Of Chicago 5801 S Ellis Ave Chicago, Il 60637

Grant 2P30CA014599-289023 from National Cancer Institute, IRG: NCI

Abstract: The realization that defined tumor antigens can serve as targets for recognition by T lymphocytes has motivated attempts at developing and optimizing immunologic therapies for the treatment of cancer. This excitement has been reinforced by the success of two humanized monoclonal antibodies, Herceptin and Rituxan, for the treatment of breast cancer and lymphoma, respectively, as well as the approval of two cytokines, interleukin-2 and interferon-alpha, for the treatment of melanoma. The development of new immune-based therapies, such as cancer vaccines and novel cytokines, and the elucidation of the mechanism of action of currently available treatments, require careful monitoring of scientific endpoints to determine the optimal biologically active dose and schedule of these agents. The purpose of the Human Immunologic Monitoring Facility is to perform such assays in the context of clinical trials in cancer patients. This service enables a range of clinical cancer researchers, who may not themselves have the expertise or laboratory commitment to carry out these assays, to measure Immunotherapy Group as it develops cancer treatment strategies involving vaccines, monoclonal antibodies, cytokines, and cell-based therapies. immunologic endpoints in participating study subjects. Assays currently performed include measurement of T cell cytokine production, proliferation, and cytolytic activity; serum cytokine content; flow cytometric analysis of hematopoietic and T cell subsets; induced intracellular cytokines by flow cytometry; and RT-PCR analysis of tumor antigen gene expression. Preparation of peptide-based cancer vaccines is also performed. New assays are under development that will incorporate the latest techniques and enable important new scientific questions to be asked in the clinical setting. In sum, this facility provides clinical trial laboratory support for our Cancer

Keywords: biomedical facility, immunologic assay /test, neoplasm /cancer immunology, T lymphocyte, dosage, flow cytometry, neoplasm /cancer immunotherapy, tumor antigen, clinical research, human tissue

Project start date: 2002-05-15

Project end date: 2007-03-31

T Cell Responsiveness And Homeostasis In Anti-Tumor

Thomas F Gajewski, Associate Professor
University Of Chicago 5801 S Ellis Ave Chicago, Il 60637

Grant 1R01CA118153-01A1 from National Cancer Institute, IRG: TTT

Abstract: The progressive growth of antigen-expressing tumors despite the presence of tumor antigen-specific CD8+ T cells is a central problem in the field of anti-tumor immunity. Although one hypothetical mechanism of escape is T cell anergy, this process in the tumor setting is poorly defined. Using a TCP transgenic T cell model that allows recovery of sufficient numbers of T cells for analysis ex vivo, we have recently observed that tumors do induce antigen-specific hyporesponsiveness of anti-tumor CD8+ T cells and not of irrelevant T cells, consistent with a form of T cell anergy. We also have obtained preliminary data that tumor-induced T cell hyporesponsiveness does not occur when lymphopenia-induced homeostatic proliferation is permitted. Understanding the mechanism of the induction and reversal of T cell anergy should have great impact in our ability to maintain the functionality of tumor antigen-specific T cells in vivo, and enable the development of interventions that can be translated to the clinic. The first specific aim is to determine the mechanism of T cell-intrinsic hyporesponsiveness of TCR transgenic T cells in tumor bearing mice. TCR Tg T cells will be analyzed ex vivo for deficiencies in signal transduction events triggered by TCR/CD28 stimulation, and for molecular alterations using gene expression profiling. CAR Tg T cells and adenoviral vectors will be utilized to examine the causal relationship between observed changes and T cell dysfunction. The second specific aim is to explore and understand the mechanism by which T cell responsiveness is restored through homeostatic proliferation in vivo. Homeostatic signals will be delivered to anergic T cells by manipulating the host (transferring into RAG2-/- mice), manipulating the tumor (transfecting to express IL-7 and/or IL-15), or by manipulating the T cells (transduction with constitutively active Stat5). T cells will be analyzed for functional and biochemical properties, and alterations will be correlated with tumor rejection capability. The third specific aim is to apply knowledge of maintenance of T cell responsiveness out of this reductionist model toward rejection of established tumors in normal mice. T cell subsets from normal C57BL76 mice will be manipulated to affect homeostatic factors and tested for rejection of B16 melanoma transfected to express the model antigen SIY. T cell transduction and tumor transfectants will be examined using factors identified to be useful from the TCR Tg model and mechanisms of improved tumor control will be dissected.

Keywords: homeostasis, neoplasm /cancer

Project start date: 2006-09-28

Project end date: 2011-07-31

1R01CA118153-01A1 (2006): $268816

Multi-Peptide/IL 12 Melanoma Vaccine

Thomas F Gajewski, Associate Professor
University Of Chicago 5801 S Ellis Ave Chicago, Il 60637

Grant 5R01CA090575-04 from National Cancer Institute, IRG: CONC

Abstract: The existence of tumor antigens that can serve as targets for recognition by specific cytolytic T lymphocytes (CTL) suggests that a successfully generated and executed immune response should be capable of supporting tumor rejection. The failure of human tumors to be rejected spontaneously in vivo, therefore, must be a consequence either of a lack of induction of a potent tumor antigen-specific CTL response or of resistance at some level to the immune response induced. Our preclinical and clinical studies have indicated that immunization with tumor antigen peptide-pulsed PBMC + IL-12 can induce CTL responses in vivo. However, some patients do not experience tumor regression despite having detectable CTL in the blood. These observations suggest that downstream resistance to effector CTL may be dominant. Understanding mechanisms of tumor escape from immune-mediated destruction in every treated patient will guide the next level of intervention required to increase the clinical response to immune-based therapies. In Specific Aim 1 we will determine whether immunization with 4 melanoma peptides gives a higher clinical response rate than single peptide immunization has produced. This is based on the hypothesis that selection for antigen-loss variants should be less frequent. In Specific Aim 2 we will examine whether the addition of very low-dose IL-2 following each vaccine will increase the frequency of specific CD8+ T cells using tetramer analysis. This is based on the recent observation that very low-dose IL-2 can support the survival of adoptively transferred human CTL clones. In Specific Aim 3 we will study intensively the tumor microenvironment for mechanisms of immune escape. Expression of tumor antigen genes, class I MHC, and TAP on tumor cells will be examined, and expression of positive and negative regulatory cytokines by real-time RT-PCR will be assessed. Finally, gene array screens will be performed with the aim of identifying gene expression patterns that correlate with susceptibility versus resistance to the therapeutic effect of peptide vaccination. Collectively, these results will optimize immunization with melanoma peptide-pulsed PBMC + rhlL-12 and identify the prevalence of various mechanisms of tumor resistance to vaccine efficacy. Understanding these mechanisms will illuminate the next level of intervention to develop to increase the clinical response to the immunotherapy of melanoma.

Keywords: cytotoxic T lymphocyte, drug screening /evaluation, interleukin 12, leukocyte activation /transformation, melanoma, metastasis, neoplasm /cancer immunotherapy, neoplasm /cancer vaccine, synthetic vaccine, MHC class I antigen, active immunization, cytokine, gene expression, genetic susceptibility, human therapy evaluation, interferon gamma, interleukin 2, biopsy, clinical research, enzyme linked immunosorbent assay, flow cytometry, human subject, immunocytochemistry, microarray technology, patient oriented research, polymerase chain reaction

Project start date: 2003-05-01

Project end date: 2007-04-30

5R01CA090575-04 (2006): $265071

5R01CA090575-03 (2005): $271450

5R01CA090575-02 (2004): $271450

1R01CA090575-01A1 (2003): $271450

Deletion Of Inhibitory Genes In Post-thymic T Cells To Study Immune Tolerance

Thomas F Gajewski, Associate Professor
Pathologyuniversity Of Chicago

Grant 1R21AI079373-01 from National Institute Of Allergy And Infectious Diseases, IRG: ZRG1

Abstract: The conditional deletion of targeted genes using the Cre/lox system has provided an invaluable tool for understanding the functional role of specific gene products in selected cell lineages in vivo. For studies of T cell biology, mice bearing targeted alleles are typically intercrossed with transgenic mice expressing Cre driven by the Lck, CD2, or CD4 promoters. Clear functional roles of specific signaling molecules have been established using such systems in the T cell lineage. However, as these promoters are active in developing thymocytes, the most striking phenotypes have been obtained in that compartment. In addition, because thymic selection often becomes perturbed in such mutant mice, the results of studies in peripheral T cells can be difficult, or even impossible, to interpret. Using mice transgenic for the Coxsackie and adenovirus receptor (CAR) in the T cell lineage, we recently have developed a strategy for conditional deletion of genes in post- thymic T cells using a Cre adenovirus. This tool has now allowed us to explore for the first time the deletion of specific genes of interest directly in peripheral T cells. We propose to apply this technique to gain a better understanding of the role of the lipid phosphatase PTEN in T cell biology. Conditional PTEN-deficient mice on an Lck-Cre background develop autoimmunity and T cell lymphoma. We hypothesize that these phenotypes may be a result of PTEN deletion in the thymus and might not occur when PTEN is deleted in post-thymic T cells, in which case heightened T cell immunity may theless be seen. In the first Specific Aim, control and PTEN-deleted T cells will be analyzed for potentiation in IL-2 production, proliferation, and survival. Effects on differentiation into effector cell subsets, and regulation of downstream signaling events, also will be investigated. In the second Specific Aim, control and PTEN-deleted T cells will be transferred in vivo to investigate the potential for autoimmunity and lymphoma development. Whether augmented immune responses to vaccination or against syngeneic tumors occurs also will be examined. Ultimately, these experiments will allow assessment of whether pharmacologic targeting of PTEN as a future potential immunopotentiating intervention is of interest for further development. PUBLIC HEALTH RELEVANCE A strategy will be explored for genetic deletion of the negative regulatory molecule PTEN directly in peripheral T cells. Whether PTEN-deleted T cells generate improved immune responses to vaccines or against tumors, without causing autoimmunity or leading to lymphoma, will be tested. If successful, the results of this project will support development of drugs to inhibit PTEN function with the ultimate aim of improving host immune responses against cancers or chronic infections

Project start date: 2008-07-01

Project end date: 2010-06-30

Countering Immune Resistance In The Melanoma Tumor Microenvironment

Thomas F Gajewski, Associate Professor
Pathologyuniversity Of Chicago
5801 S Ellis Ave
chicago, Il 60637

Grant 5R01CA127475-02 from National Cancer Institute, IRG: ZRG1

Abstract: Recent observations have indicated that features of the melanoma tumor microenvironment likely determine whether tumor regression versus resistance occurs in response to a successfully generated anti-tumor T cell response. Our preliminary gene expression profiling data on the melanoma tumor microenvironment from patients with advanced disease have suggested two categories of downstream defects failure to recruit activated T cells into metastatic sites, and presence of immunosuppressive mechanisms in the microenvironment of tumors that have indeed recruited T cells. T cell trafficking has been associated with expression of specific chemokines within tumor sites. Identified immune resistance mechanisms include expression of the inhibitory ligand PD-L1 on the tumor cells themselves, the presence of FoxP3+ regulatory T cells, the tryptophan-catabolizing enzyme IDO expressed by dendritic-like cells and endothelial cells, and the anergy-promoting conditions of having poor B7 expression by APC populations. An additional observation has linked high levels of Notch signaling in melanoma tumors with resistance to immunotherapy and poor T cell recruitement, thus offering a potential link between tumor cell biology and establishment of features of the surrounding microenvironment. These observations have crystalized into the following Specific Aims 1. To examine the relative contribution of PD-1, regulatory T cells, IDO, and anergy in limiting immune-mediated tumor regression in a mouse preclinical model 2. To identify cell types producing specific chemokines in the tumor microenvironment and determine the role of selected chemokines in T cell recruitment; and 3. To investigate the role of Notch signaling in melanoma tumor cells in establishing the tumor microenvironment and mediating resistance to T cell-mediated killing. The ultimate goal of this work is to develop strategies to facilitate the effector phase of the anti-tumor immune response by overcoming limitations within the melanoma tumor microenvironment, thus identifying approaches with potential for future clinical application

Keywords: cell, melanoma, neoplasm /cancer anergy, base, cell biology, cell type, chemokine, clinical trial, concept, conditioning, environment, enzyme, gene, gene expression, gene expression profiling, human, immune response, immunity, immunosuppression, immunosuppressive, immunotherapy, ligand, lighting, metastasis, model, phenotype, play, role, success, tryptophan, vaccine, xenotransplantation clinical research

Project start date: 2007-04-01

Project end date: 2012-01-31

Regulatory Signals To Promote Antitumor Immunity

Thomas F Gajewski, Associate Professor
University Of Chicago 5801 S Ellis Ave Chicago, Il 60637

Grant 1P01CA097296-01A10003 from National Cancer Institute, IRG: NCI

Abstract: Recent evidence has suggested that qualitative aspects of an anti-tumor T cell response can influence whether tumor rejection can be achieved. However, quantitative aspects of T cell activation also are likely to be critical. Intrinsic to normal T cell responses are multiple negative regulatory pathways that mediate contraction of a clonally expanded T cell pool and subsequently provide inhibitory signals for T cell re-activation. It is reasoned that these mechanisms limit the ability of antigen-expressing tumors to become rejected spontaneously, and likewise preclude effective elimination of established tumors via attempts at immunotherapeutic intervention. A central hypothesis of this proposal is that interfering with or countering these negative regulatory influences on T cell activation may improve that magnitude or duration of an anti-tumor T cell response and thus lead to more efficient tumor elimination. In Specific Aim 1, we will examine the role of the putative negative regulatory receptors Fas, CTLA4, and PD-1 on limiting the efficacy of anti-tumor CD8 + T cell response in vivo. These studies will utilize 2C TCR transgenic/RAG-/- mice interbred with mice deficient in these receptors, alone and in combination. Adoptive transfer of graded numbers of mutant T cells into recipient mice either before tumor challenge or after implantation of a pre-established tumor will enable a quantitative assessment of the contribution of each receptor to be determined. In Specific Aim 2, we will manipulate intracellular signaling pathways to counter death pathways or signaling defects that have been observed in T cell from tumor-bearing mice. Introduction of the anti-apoptotic molecules Bcl-xL and p35, or of CA mutants of the signaling molecules Lck, Ras, or IKKbeta3, will be achieved utilizing adenoviral vectors and CAR transgenic/2C/RAG2 -/- T cells. Adoptive transfer will again be performed and effects on T cell functions and anti-tumor efficacy will be determined. In Specific Aim 3, we will apply new information learned using the TCR transgenic system toward preclinical models of tumor antigen vaccines and adoptive T cell therapy in normal mice. Soluble receptor antagonists, or retroviral transduction of T cells, will be performed to improve immunotherapy of mice in protection assays, treatment of established tumors, and also in mice acquiring spontaneous HPV E7-expressing tumors. The results of these studies will pave the way for future clinical translation of the most effective of these approaches.

Keywords: biological signal transduction, cellular immunity, cytotoxic T lymphocyte, immune tolerance /unresponsiveness, immunoregulation, leukocyte activation /transformation, neoplasm /cancer immunology, neoplasm /cancer remission /regression, CD28 molecule, T cell receptor, apoptosis, cytokine receptor, lymphocyte proliferation, neoplasm /cancer immunotherapy, neoplasm /cancer vaccine, nuclear factor kappa beta, protein tyrosine kinase, protooncogene, serine threonine protein kinase, tumor antigen, clinical research, gene targeting, genetic transduction, laboratory mouse, monoclonal antibody, transfection /expression vector, transgenic animal

Project start date: 2003-06-25

Project end date: 2008-03-31

Technologies To Block Gene Expression In Normal T Cells

Thomas F Gajewski, Associate Professor
University Of Chicago 5801 S Ellis Ave Chicago, Il 60637

Grant 5R21AI053886-02 from National Institute Of Allergy And Infectious Diseases, IRG: ALY

Abstract: Our preliminary results from the R01 grant that this application intends to support have revealed multiple genes that are differentially expressed in naive versus effector CD8+ T cells. The methodology of utilizing T cells transgenic for the Coxsackie and adenovirus receptor (CAR) transduced with adenoviral vectors has generated a powerful model for introducing wild type or mutant genes into resting post-thymic T cells for mechanistic studies. However, there are several attractive candidate genes the expression of which we would like to down-regulate in normal resting T cells, either in the naive state or following differentiation However, technologies to achieve this are lacking. The overall goal of the current proposal is develop and implement two strategies to eliminate expression of genes in normal post-thymic T cells at any desired differentiation state. Each approach takes advantage of the high transduction efficiency achieved with adenoviral vectors and CARTg T cells. In Specific Aim 1 we will develop and apply antisense adenoviral vectors to down-regulate expression of specific genes in CARTg T cells. The glucose transporter GLUT1 and the cytoskeletal protein calpactin/Annexin II will be studied first. In Specific Aim 2 we will develop and implement a system for conditional gene targeting using a Cre adenovirus and CARTg T cells. A LoxP/LacZ reporter mouse will be used as proof of concept, and conditional targeting constructs will be produced for GLUT1 and calpactin/Annexin II. Collectively, these technologies will expand greatly our ability to study the function of specific gene products in controlling functions of normal peripheral CD8+ T cells, with potential application toward a multitude of problems in T cell biology.

Keywords: Adenoviridae, annexin, antisense nucleic acid, cytotoxic T lymphocyte, gene expression, gene targeting, glucose transporter, leukocyte activation /transformation, technology /technique development, transfection /expression vector, cell differentiation, complementary DNA, genetic model, genetic regulation, recombinant virus, reporter gene, ubiquitin, biotechnology, laboratory mouse, transgenic animal

Project start date: 2002-09-30

Project end date: 2004-08-31

5R21AI053886-02 (2003): $147460

Biochemical/Molecular Changes Upon Naive T Cell Priming

Thomas F Gajewski, Associate Professor
Pathologyuniversity Of Chicago
5801 S Ellis Ave
chicago, Il 60637

Grant 1R01AI047919-01A1 from National Institute Of Allergy And Infectious Diseases, IRG: IMB

Abstract: Naive CD8+ T lymphocytes lack effector function and undergo a differentiation process to exit the naive state and to acquire a primed effector phenotype. Preliminary results indicate that this transition is a complex and highly regulated process, involving receptor-mediated signal transduction, cell cycle progression, chromatin remodeling, and changes in gene expression. Evidence suggests that the naive state may be actively maintained, raising the possibility of a genetic program to place a brake on differentiation. In contrast, primed T-cells gain effector function, as well as altered signal transduction pathways and a more dynamic actin cytoskeleton, suggesting that new genes become expressed to mediate these processes. The overall goal of this application is to identify the molecular phenotype of naive and primed effector CD8+ T-cells, and to study the functional role of identified molecules in normal T-cell populations. The principal model will utilize 2C TCR transgenic mice crossed to a Rag2 background, to extinguish endogenous TCR gene rearrangement and ensure a naive phenotype. Mechanistic experiments in normal, post-thymic, T-cells will be enabled by a novel Coxsackie and adenovirus receptor (CAR) transgenic mouse and adenoviral transduction. The first specific aim is to identify genes and proteins that are differentially expressed between naive and primed effector CD8+ T-cells, by I) examination of candidates by Western blotting, ii) analysis of expression of defined genes using Affymetrix microarrays, and iii) identification of unknown genes by subtractive library cloning. The second specific aim is to optimize the CAR transgenic mouse system for transduction of normal, post-thymic T-cells. Transgenic mice expressing CAR have been constructed using an LckICD2 promoter/enhancer cassette. Optimization of adenoviral transduction will be performed, and interbreeding with 2C/Rag2-/- mice will be carried out for studies of naive and primed CD8+ T-cells. The third specific aim is to analyze the function of differentially expressed signaling molecules and transcription factors linked to MAP kinase pathways in normal T-cells using CAR transgenic mice. Preliminary results have shown that several proteins involved in MAP kinase signaling become markedly up-regulated upon T-cell priming. Wildtype and mutant Ras, FosB, MKK7, JNK1, JNK2, and other candidates will be introduced into naive and primed CAR transgenic T-cells. Effects on early T-cell differentiation, as well as on apoptosis, TCR sensitivity, CD28 independence, and magnitude of cytokine production, will be examined. The fourth specific aim is to investigate the functional requirement for modulators of actin cytoskeleton dynamics in normal T-cells. Preliminary results have revealed that expression of several molecules associated with cytoskeletal dynamics is increased tremendously upon differentiation from the naive to the primed effector state. Wildtype and mutant a-Pak, Raci, and other candidates will be introduced into primed effector T-cells, and potential effects on TCR capping, conjugate formation, cell spreading, cytolysis, and cytokine production will be investigated. In addition, gelsolin4 -/- mice will be examined for functional defects in the T-cell compartment. The results of these experiments will provide invaluable information regarding the molecular and biochemical changes that accompany T-cell differentiation, pointing the way to new potential targets for manipulating immune responses in vivo

Keywords: T lymphocyte, cell differentiation, gene expression, protein biosynthesis CD28 molecule, T cell receptor, apoptosis, biological signal transduction, cytokine, cytoskeletal protein, protein kinase, transcription factor Adenoviridae, Coxsackievirus, laboratory mouse, microarray technology, transfection, transgenic animal

Project start date: 2001-04-01

Project end date: 2006-03-31

1R01AI047919-01A1 (2001): $279523

5R01AI047919-05 (2005): $282549

5R01AI047919-04 (2004): $282732

5R01AI047919-03 (2003): $278887

Countering Immune Resistance In The Melanoma Tumor Microenvironment

Thomas F Gajewski, Associate Professor
Pathologyuniversity Of Chicago, 5801 S Ellis Ave, Chicago, Il 60637

Grant 1R01CA127475-01 from National Cancer Institute, IRG: ZRG1

Abstract: DESCRIPTION () Recent observations have indicated that features of the melanoma tumor microenvironment likely determine whether tumor regression versus resistance occurs in response to a successfully generated anti-tumor T cell response. Our preliminary gene expression profiling data on the melanoma tumor microenvironment from patients with advanced disease have suggested two categories of downstream defects failure to recruit activated T cells into metastatic sites, and presence of immunosuppressive mechanisms in the microenvironment of tumors that have indeed recruited T cells. T cell trafficking has been associated with expression of specific chemokines within tumor sites. Identified immune resistance mechanisms include expression of the inhibitory ligand PD-L1 on the tumor cells themselves, the presence of FoxP3+ regulatory T cells, the tryptophan-catabolizing enzyme IDO expressed by dendritic-like cells and endothelial cells, and the anergy-promoting conditions of having poor B7 expression by APC populations. An additional observation has linked high levels of Notch signaling in melanoma tumors with resistance to immunotherapy and poor T cell recruitement, thus offering a potential link between tumor cell biology and establishment of features of the surrounding microenvironment. These observations have crystalized into the following Specific Aims 1. To examine the relative contribution of PD-1, regulatory T cells, IDO, and anergy in limiting immune-mediated tumor regression in a mouse preclinical model 2. To identify cell types producing specific chemokines in the tumor microenvironment and determine the role of selected chemokines in T cell recruitment; and 3. To investigate the role of Notch signaling in melanoma tumor cells in establishing the tumor microenvironment and mediating resistance to T cell-mediated killing. The ultimate goal of this work is to develop strategies to facilitate the effector phase of the anti-tumor immune response by overcoming limitations within the melanoma tumor microenvironment, thus identifying approaches with potential for future clinical application.

Project start date: 2007-04-01

Project end date: 2012-01-31

1R01CA127475-01 (2007): $291650

Molecular Dissection Of T Cell Anergy

Thomas F Gajewski, Associate Professor
University Of Chicago 5801 S Ellis Ave Chicago, Il 60637

Grant 5R21AI059818-02 from National Institute Of Allergy And Infectious Diseases, IRG: ZAI1

Abstract: Although immunologic tolerance is controlled in part through thymic deletion, mechanisms of peripheral tolerance must be in place to avoid autoreactive aggression of T cells that survive into the periphery. One proposed mechanism of peripheral tolerance is the process of clonal anergy, a hyporesponsive state resulting from engagement of the TCR in the absence of costimualtory signals. Although anergy has been studied for many years, the precise molecular mechanism to explain the functional defects in anergic T cells remains unclear. We previously demonstrated defective TCR-induced Ras/MAP kinase signaling in anergic T cells. Using a novel adenoviral transduction system, we recently have shown that constitutively active (CA) Ras restores IL-2 production and MAP kinase activation in anergized T cells, arguing that defective Ras activation is causally linked to hyporesponsiveness. To understand the mechanism for blunted Ras activity in anergic cells we performed a gene array screen that has identified several attractive candidates. One of these is diacylglycerol kinase (DGK). However, whether upregulated DGK is sufficient to explain all the features of anergy is not clear, and the role of other potential candidates is not known. In Specific Aim 1, whether upregulation of DGK is sufficient to explain all features of the anergic state will be investigated. These experiments will be facilitated by the use of Coxsackie and adenovirus receptor (CAR) transgenic T cells and adenoviral vectors encoding wildtype and kinase-dead DGK isoforms. In Specific Aim 2, other attractive gene candidates will be considered, focusing on Schlafen and CRTAM. Adenoviral constructs will be made to study whether normal T cell activation will be suppressed. Monoclonal antibodies will be generated against CRTAM to study regulation of protein expression and effects of ligation or blockade on T cell function. New candidates will also be identified using EST arrays. Ultimately, a complete understanding of the anergic state on the molecular level should guide the development of novel pharmacologic therapies to promote or reverse peripheral tolerance in vivo.

Keywords: T cell receptor, T lymphocyte, anergy, diacylglycerol, gene expression, genetic regulation, leukocyte activation /transformation, protein kinase, Adenoviridae, Coxsackievirus, laboratory mouse, microarray technology, monoclonal antibody, transfection /expression vector

Project start date: 2004-07-01

Project end date: 2006-06-30

5R21AI059818-02 (2005): $223710

1R21AI059818-01 (2004): $223710

MAGE-3 PLUS IL-12 IMMUNIZATION IN METASTATIC MELANOMA

Thomas F Gajewski, Associate Professor
University Of Chicago 5801 S Ellis Ave Chicago, Il 60637

Grant 5M01RR000055-420709 from National Center For Research Resources, IRG:

Keywords: combination cancer therapy, human therapy evaluation, interleukin 12, melanoma, metastasis, neoplasm /cancer chemotherapy, neoplasm /cancer immunotherapy, tumor antigen, clinical trial phase I, clinical research, human subject